MRM/PRM Targeted Exosomal Proteomic Quantitative Service

The complexity of exosomal samples limits non-targeted proteomics quantification techniques, low abundance peptide signals are easily suppressed by high abundance, therefore making it difficult to detect low abundance exosomal peptides, with low sensitivity and randomness, and low reproducibility of quantification. To overcome this shortcoming, Creative Biolabs offers a high-throughput and highly accurate MRM/PRM targeted exosomal proteomic quantitative service that can validate these exosome-based markers in large biological sample sizes based on differential proteomic data.

Introduction of MRM/PRM Targeted Exosomal Proteomic Quantitative

Targeted quantitative proteomics is a technique that takes full advantage of the characteristics of mass spectrometers to perform multiple screening of ions entering the MS, and then target the fragmented ions of selected target exosomal peptides for targeted detection. Currently, with the development of targeted quantitative proteomics technology, two main technical routes have been developed, MRM (Multiple Reaction Monitoring) and PRM (Parallel Reaction Monitoring).

MRM is a technique developed for targeted quantitative analysis of multiple specific proteins/peptides/small molecules in complex samples using the qualitative multiplex ion screening capability of the triple-quadrupole mass spectrometer. The MS analysis of MRM proceeds through three stages: 1) MS screening for parent ions that are specific to the target molecule; 2) collision fragmentation of these parent ions to remove interference from other ions; 3) Acquisition of mass spectrometric signals for selected specific MS/MS2 ions only.

PRM with a high resolution/high precision mass spectrometer has the advantage of being an easy-to-operate derivative of MRM without prior determination of ion pairs and optimization of collision energies. First, the parent ion of the target peptide is selectively detected using a four-stage mass analyzer. Subsequently, the parent ions are fragmented in a collision cell. Finally, all fragmentation information within the selected parent ion window is detected in a secondary mass spectrometer using an Orbitrap analyzer for quantitative analysis of the target exosomal peptides.

Fig.1 Schematic representation of PRM (top) and MRM (bottom) as performed on Q-Orbitrap and QQQ instrumentation respectively. (Zhou & Yin, 2016)

Features and Applications of MRM/PRM

Targeted proteomics quantification is an alternative to antibody-based protein quantification techniques for signaling pathway detection, tumor marker studies, and post-translational modification studies. Both MRM and PRM are better for the targeted quantification of data from non-targeted proteomic screens.

MRM is a targeted large-molecule protein detection technique based on a dedicated platform for targeted molecular detection, the triple quadrupole. It excludes the interfering effects of other ions as much as possible, improves the signal-to-noise ratio of targeted peptides, and the sensitivity and reproducibility of qualitative and quantitative detection of target peptides, which is considered to be suitable for high-throughput monitoring of marker proteins based on MS for protein quantification. MRM technique can also be used for absolute quantification of target proteins by adding known levels of isotopically labeled peptides to the sample as an internal reference. MRM technique is particularly suitable for the quantitative detection of specific small groups of target exosomal proteins in high-volume samples.

PRM is a targeted large-molecule protein detection technique developed by relying on the high-resolution performance of mass spectrometers. It is characterized by a proteome screening and validation that can be achieved on a single mass spectrometer, easy operation, low detection cost, and a large number of ions that can be targeted for monitoring. However, due to the accompanying nanoliter liquid phase system, its instrumentation is a bit less stable. When the number of peptides to be analyzed is too large, the MS acquisition parameters need to be finely adjusted, otherwise, the accuracy and precision of the quantitative data will be affected. Therefore, PRM is generally completed within a short period after the completion of non-targeted proteomics and before the next round of maintenance of the mass spectrometer. PRM is suitable for the experimental needs of small-batch exosomal protein samples with more protein-targeted quantitative assays.

MRM/PRM Targeted Exosomal Proteomic Quantitative Service at Creative Biolabs

Based on the target protein information you provide for the desired study, we provide one-stop MRM/PRM exosome quantitative protein analysis services, including MRM/PRM method establishment and optimization, specific peptide selection, isotope-labeled peptides, MS analysis, raw data analysis, and bioinformatics analysis. Multiple aspects of bioinformatics analysis are available, such as

Spectrogram analysis, and protein data quality assessment.
PCA analysis of multi-component samples.
Functional annotation of identified proteins: GO functional annotation, KEGG functional annotation, and COG functional annotation.
Statistical analysis of differentially expressed proteins: Venn diagram, Volcano Plot.
Cluster analysis of differentially expressed proteins: hierarchical clustering, K-means clustering.
Differential protein interaction analysis: protein interaction network analysis based on the STRING database.

Fig.2 MRM/PRM targeted exosomal proteomic quantitative service workflow.

Creative Biolabs offers MRM/PRM targeted exosomal proteomic quantitative services that enable selective detection and precise quantification of exosomal proteins through high-resolution, high-precision MS. Please contact us to get a quote.

Reference

Zhou, J.; Yin, Y. Strategies for large-scale targeted metabolomics quantification by liquid chromatography-mass spectrometry. Analyst. 2016, 141(23): 6362-6373.

Plant Exosome Isolation